Diphosphosinositol polyphosphates and energy metabolism: assay for ATP/ADP ratio

Methods in Molecular Biology
Andreas NagelChristopher Illies

Abstract

Several inositol compounds undergo rapid cycles of phosphorylation and dephosphorylation. These cycles are dependent on ATP and energy metabolism. Therefore, interfering with the cellular energy metabolism can change the concentration of rapidly turning over inositols. Many pharmacological inhibitors, apart from their intended action, also affect the energy metabolism of the cells and lower ATP. This can unspecifically influence rapidly turning over inositol phosphates. Thus, the ATP concentration should be checked when reduced inositol phosphates are observed after application of pharmacological inhibitors. A luminescence-based assay for the measurement of ATP and ADP is described. ATP is measured luminometrically using firefly luciferase. Detection of ADP is performed in a two-step enzymatic procedure: (1) The sample ATP is degraded to AMP and (2) ADP is phosphorylated to ATP, which can then be measured luminometrically. This method gives a better signal-to-noise ratio than other methods that do not degrade the sample ATP, but convert ADP directly to ATP and then measure the sum of ATP plus ADP.

Citations

Feb 23, 2013·Pharmacological Reviews·Christopher J Barker, Per-Olof Berggren
Sep 4, 2015·Journal of Biosciences·Swarna Gowri Thota, Rashna Bhandari
Jan 1, 2017·Journal of the Indian Institute of Science·Akruti ShahRashna Bhandari
Oct 1, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Mirko MininiMariano Bizzarri

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